| Budget Amount *help |
¥15,000,000 (Direct Cost: ¥15,000,000)
Fiscal Year 2003: ¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 2002: ¥7,800,000 (Direct Cost: ¥7,800,000)
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| Research Abstract |
In order to understand molecular mechanisms of tertially structure formation and quality control, we undertook two types of research, studies using biochemical and bioimaging analysis. As major outcome of the former investigation, we found that EDEM, an ERAD stimulating lectin-like molecule, forms tight and stable complex with calnexin through a transmembrane region, and that substrates glycoproteins are transferred from calnexin to EDEM upon processing of N-linked oligosaccharides. Previously we identified EDEM as a molecular which promotes proteasomal disposal of abberant glycoproteins. Futher, we analyzed the disulfide exchanging reactions by using as a model molecule fibrinogen, which forms complicated tertially strucuture and contains numerous number of disulfide bonds. While studing the details of the nascent chain folding, we found that the ER is equipped with a novel mechanism which enables to export selectively the terminally misfolded aggregates.
As an approach employing bioim
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aging analysis, we performed a series of analysis using fluorescent correlation spectroscopy (FCS) which measures diffusional motion of fluorescent molecules in a very small confocal volume generated by laser confocal microscopy. FCS allows us to study movements of proteins at a single molecule level in living cells. As a result, we found that intense supression of random movements relates to maintenance of foldability at high temperature where folding tends to be failed. Also, we showed that it is possible to use GFP and mRFP1 to study binding of two proteins in living cells using FCCS technique (fluorescence cross correlation techinique). However, this study also revealed that there is a limit when using mRFP1 for this pourpose because of low brightness and high flickering properties. Furthermore, by studing the dynamics of glycoproteins in details, we found that it is possible to visualize mobility of a single proteins in the ER network and the ER contains a mechanisms to regulate random motion of glycoproteins.
Unfortunately, we were not succeed in identifing proteins which could recognize pro domains of secretory proteins although we were able to isolate several candidate proteins as a receptor but not eble to prove their function in the context. For this purpose, further studies are required. Less
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